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Development of a disease model targeting on a newly discovered protein, parchorin, specifically expressed in water-secreting cells

针对新发现的蛋白质 parchorin 开发疾病模型，该蛋白质在泌水细胞中特异性表达

基本信息

批准号：
13557220
负责人：
URUSHIDANI Tetsuro
金额：
$ 7.55万
依托单位：
National Institute of Health Sciences (2003)The University of Tokyo (2001-2002)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13557220/
关键词：
parchorin knockout mouse CLIC family protein

项目摘要

We recently cloned a new protein, parchorin, from rabbit choroid plexus. Parchorin belongs to Chloride Intracellular Channel (CLIC) family and is specifically expressed in cells participating water movement in various tissues. In the present study, we tried to produce a parchorin-knock out mouse to utilize it as a disease model. Based on its rabbit sequence, RT-PCR was performed for mRNA obtained from mouse choroid plexus. Although we cloned a new family member of CLIC, it did not seem to be parchorin. Meanwhile, one of our colleague isolated human CLIC5A and its splicing variant CLIC5B. From its sequence and tissue distribution, our cloned sequence was considered to be a mouse homologue of CLIC5A. We then purified parchorin protein to get amino acid sequence, and determined the whole sequence of mouse parchorin utilizing, the results of mouse genome project as well. Based on the published genomic data, construction of targeting vector for substituting parchorin exon 1 was performed. We employed a strategy to eliminate neomycin resistant gene for selection as well as to enable a conditional knock out using both Cre/lox and FLP/Frt systems. Using established methods, recombinant mouse ES cells and subsequently chimera mice were obtained. At the end of the project, hetero-mice were born. We are now planning to analyze their pathophysiology after the knockout mice are born.

我们最近从兔子脉络丛中克隆了一种新蛋白质parchorin。Parchorin属于细胞内氯离子通道（CLIC）家族，在多种组织中特异性表达于参与水分运动的细胞中。在本研究中，我们试图建立一个parchorin基因敲除小鼠，利用它作为一个疾病模型。根据兔的序列，从小鼠脉络丛中获得mRNA，进行RT-PCR。虽然我们克隆了一个新的CLIC家族成员，但它似乎不是parchorin。与此同时，我们的一位同事分离了人CLIC 5A及其剪接变体CLIC 5 B。从其序列和组织分布来看，我们克隆的序列被认为是CLIC 5A的小鼠同源物。利用小鼠基因组计划的结果，对小鼠parchorin蛋白进行了纯化和氨基酸序列测定。基于已发表的基因组数据，进行了用于替换parchorin外显子1的靶向载体的构建。我们采用了一种策略，以消除新霉素抗性基因的选择，以及使条件敲除使用Cre/lox和FLP/Frt系统。使用已建立的方法，获得重组小鼠ES细胞和随后的嵌合体小鼠。在项目结束时，异基因小鼠诞生了。我们现在正计划在基因敲除小鼠出生后分析它们的病理生理学。